Polyphenol Extraction Process

ABSTRACT

The invention relates to a process for extracting one or more polyphenols from fruits such as apples and to uses of said extracts in the treatment or prophylaxis of cardiovascular disease, colon cancer and digestive health.

The invention relates to a process for extracting one or morepolyphenols from fruits such as apples and to uses of said extracts inthe treatment or prophylaxis of cardiovascular disease, colon cancer anddigestive health.

Cardiovascular disease (CVD) and cancer are two leading causes of deathworldwide.

In the European Union, CVD is the main source of morbidity andmortality, costing 169 billion Euros annually (Leal et al. (2006) Eur.Heart J. 27(13): 1610-9), whilst in the US, the American HeartAssociation has estimated that 71,300,000 Americans have one or moreforms of CVD.

According to the World Health Organisation, cancer kills about 7.6million (or 13%) people worldwide every year. In particular, cancers ofthe lung, stomach, liver, colon and breast are responsible for over halfthese deaths.

Epidemiological evidence seems to suggest that a diet high in fruits andvegetables offers a significant protective effect against these chronicdiseases. Much of this protective effect has been attributed to a majorclass of phytochemicals found commonly in fruits and vegetables calledpolyphenols.

Polyphenols have been referred to as nature's biological responsemodifiers because of strong experimental evidence demonstrating theirability to modify the body's reaction to allergens, viruses, andcarcinogens. They show anti-allergic, anti-inflammatory, anti-microbialand anti-cancer activity. In addition, polyphenols act as powerfulantioxidants, protecting against oxidative and free radical damage, andhelp to prevent various diseases associated with oxidative stress.

However, it has been estimated that most people do not consumesufficient quantities to obtain the health benefits offered bypolyphenols. For example, the average current US daily intake ofepicatechin is 2 to 4 times lower than the projected dose required toachieve a CVD risk reduction (Gu et al. (2004) J Nutr 134(3): 613-7;Prior and Gu (2005) Phytochemistry 66(18): 2264-80). As a result,increasing intake through the diet has emerged as an important healthgoal.

Apples can be a significant source of polyphenols, providingapproximately 10% of the projected dose required to achieve a CVD riskreduction (Gu et al. (2004) supra). However, the concentration anddistribution of polyphenols varies dramatically in each variety (seeTable 1). Indeed, many modern dessert apples can have a polyphenolcontent less than 1000 mg/kg.

Furthermore, processing of apples has been found to affect thepolyphenolic content. For example, as much as 95% of the totalpolyphenols may remain in the pomace when apple juice is extracted. Moreimportantly, procyanidins have the lowest yield in the juice (32%)(Guyot and Marnet (2003) J. Agric. Food Chem. 51(21), 6240-6247).

It is therefore an object of this invention to provide an improvedprocess for isolating polyphenols from fruits such as apples.

According to a first aspect of the invention, there is provided aprocess for extracting one or more polyphenols from a fruit whichcomprises the steps of:

(a) removing the coating of the fruit;(b) freeze-drying or freezing the fruit obtained in step (a);(c) optionally powdering the freeze-dried or frozen material obtained instep (b);(d) subjecting the material obtained in step (b) or step (c) to one ormore solvent extraction cycles;and optionally thereafter(e) isolating one or more polyphenols from the liquid extract obtainedin step (d).

In one embodiment of the invention, there is provided a process forpreparing a polyphenol-containing fraction from an apple which comprisesthe steps of:

(a) removing the natural wax coating of the apple;(b) freeze-drying or freezing the apple obtained in step (a); andoptionally thereafter(c) powdering the freeze-dried or frozen material obtained in step (b).

In a further embodiment of the invention, there is provided a processfor isolating one or more polyphenols from an apple which comprises thesteps of:

(a) removing the natural wax coating of the apple;(b) freeze-drying or freezing the apple obtained in step (a);(c) powdering the freeze-dried or frozen material obtained in step (b);(d) subjecting the powdered material obtained in step (c) to one or moresolvent extraction cycles; and optionally thereafter(e) centrifuging the liquid extract obtained in step (d).

In one embodiment, the fruit is an apple. In a further embodiment, thefruit is a “red-cut through” apple. It will be appreciated that the term“red-cut through” is well known in the art and refers to an apple havingred flesh.

When used herein, the term polyphenol refers to a group of chemicalscharacterised by the presence of more than one phenol group permolecule. In one embodiment, the polyphenols include the sub-groupflavonoids.

Over 5000 naturally occurring flavonoids have been characterised fromvarious plants. They have been classified according to their chemicalstructure and can be categorised into various sub-classes. In oneembodiment, the flavonoids include:

(a) flavanones (e.g. Hesperetin, Naringenin, Eriodictyol), which use thechemical skeleton shown in formula (I):

(b) isoflavanones (e.g. Genistein, Daidzein, Glycitein), which use thechemical skeleton shown in formula (II):

(c) flavones (e.g. Luteolin, Apigenin, Tangeritin), which use thechemical skeleton shown in formula (III):

(d) flavonols (e.g. Quercetin, Kaempferol, Myricetin, Isorhamnetin,Pachypodol, Rhamnazin), which use the chemical skeleton shown in formula(IV):

(e) flavan-3-ols (e.g. Catechin, Gallocatechin, Epicatechin,Epigallocatechin), which use the chemical skeleton shown in formula (V):

(f) flavan-3,4-diol, which use the chemical skeleton shown in formula(VI):

(g) dihydroflavonols, which use the chemical skeleton shown in formula(VII):

(h) anthocyanidins (e.g. Cyanidin, Delphinidin, Malvinidin,Pelargonidin, Peonidin, Petunidin), which use the chemical skeletonshown in formula (VIII):

(i) anthocyanins, which use the chemical skeleton shown in formula (IX):

wherein sugar may be selected from glucose, arabinose, galactose or thelike.

In one embodiment, the flavonoids include proanthocyanidins.Proanthocyanidins are biopolymers composed of flavan-3-ol subunits.Polymers are linked principally through the 4 and 8 positions and 4 and6 positions. In a further embodiment, the proanthocyanidin is a 4,8linked polymer, having the structure shown in Formula (X):

In one embodiment, the flavonoids include anthocyanidins,proanthocyanidins, flavanols, flavonols, flavones, flavanones andisoflavones. In a further embodiment, the flavonoids include flavonolsand proanthocyanidins.

In one embodiment, the flavan-3-ols have the structure shown in formula(V)a:

wherein R¹ represents hydrogen or an OH group.

In one embodiment, the flavan-3-ols have the structure shown in formula(V)b:

wherein R¹ is as defined above.

Compounds of formula (V)b wherein R¹ represents hydrogen are known asepicatechin and compounds of formula (V)b wherein R¹ represents an OHgroup are known as epigallocatechin.

In one embodiment, the flavan-3-ols have the structure shown in formula(V)c:

wherein R¹ is as defined above.

Compounds of formula (V)c wherein R¹ represents hydrogen are known ascatechin and compounds of formula (V)c wherein R¹ represents an OH groupare known as gallocatechin.

In one embodiment, the anthocyanidins include cyanidin, having thestructure shown in formula (VIII)a:

In one embodiment, the flavonoids include delphinidin, having thestructure shown in formula (VIII)b:

In one embodiment, the proanthocyanidins have the structure shown informula (X)a:

wherein R¹ is as defined above; andn=2 to 30.

In one embodiment, n>10 (i.e. 11-30). In a further embodiment, thecompounds of formula (IV) have a molecular weight in excess of 1000. Ina yet further embodiment, the compounds of formula (IV) have a molecularweight>1000 and <9000 (e.g. >3000 and <9000).

Proanthocyanidins include the subgroups of procyanidins andprodelphinidins and upon acid hydrolysis these yield cyanidin anddelphinidin respectively.

In one embodiment, the one or more polyphenols extracted by the processof the invention comprise epicatechin (i.e. a compound of formula (V)bwherein R¹ represents hydrogen).

Removal of the coating of the fruit in step (a) (e.g. removal of thenatural wax coating of an apple) advantageously reduces foaming duringthe subsequent solvent extraction cycles in step (d). In one embodiment,the removal comprises a washing step using a suitable solvent, such asethanol, methanol or acetone. The natural wax can then be recovered fromthe resultant wax/solvent extraction mixture by distillation which canadvantageously be used as a raw material for cosmetic products.

It will be appreciated that the freeze-drying or freezing step (b)typically comprises procedures known to those skilled in the art. Forexample, freeze-drying will involve pre-frozen whole apples of less than25 mm in diameter at −18° C. being dried under vacuum, ensuring that thewater is removed as vapour. This step advantageously preserves theoverall product structure and size as well as maintaining the integrityof the resultant polyphenols.

An advantage provided by the frozen samples is the production of icecrystals which rupture cellular structure and ensure easier release ofpolyphenols. Thus, freezing improves the extraction process efficiency.In addition, freeze-drying enhances the availability of catechin andepicatechin molecules, when compared with other drying methods such asoven, air or thermal drying.

The powdering step (c) comprises procedures known to those skilled inthe art. For example, powdering on a small scale may be conducted in aconventional food processor. Large scale powdering techniques maysuitably be employed in an analogous manner and will generally requireexclusion of moist air to prevent re-hydration (e.g. by use of an inertatmosphere, such as a nitrogen blanket or by purging). Powderingprevents oxidation (e.g. by polyphenol oxidase) which would result in adegradation of polyphenols and also provides small sized particles whichresults in enhancement of the subsequent extraction phase. In a furtherembodiment, powdering is carried out in the presence of liquid nitrogen,which prevents the material from macerating during the subsequentextraction cycle in step (d). The powdered apples obtained in step (c)are generally of a particle size that is beneficial for the subsequentsolvent extraction cycle, suitably between 1 and 25 mm. This aspect ofthe invention provides the significant advantage of not requiring enzymeprocessing which causes degradation of the polyphenols.

The extraction cycle in step (d) typically occurs in a continuoushorizontal counter current extractor, or a batch extractor (with solventextraction and multi-solvent extraction and downstream solventrecovery), or a counter current vertical column extractor (withdownstream liquid to liquid extraction centrifuges with wiped filmevaporators) or a semi-continuous rotary drum vacuum filter of suitabledesign or a centrifugal decanter. It will be appreciated that a countercurrent extractor is well known to those skilled in the art and couldeither be a continuous or batch counter current extractor.

Step (d) typically comprises one or more extracting solvents includingmethanol, ethanol, water, acetone and any mixture thereof. Thesesolvents help to denature plant enzymes and therefore eliminate problemsof residual enzyme activity.

In one embodiment, the extracting solvent is water. In a furtherembodiment, the water may be distilled, deionised, deaerated and heatedto a temperature between 65° C. and 80° C. (e.g. between 70° C. and 75°C., such as between 73° C. and 75° C.). The use of water as anextracting solvent provides substantial advantages, for example, it canbe used directly in the resultant foodstuff without removal and it hasno environmental impact unlike other solvents. The use of deaeratedwater provides the advantage of inhibiting oxidases which would normallycause degradation of the resultant polyphenols (reducing polyphenolyields by up to 50%). Previous approaches to this problem haveincorporated specific agents to inhibit oxidases (e.g. ascorbic acid)however this approach is of limited success because the oxidases areonly suppressed and not eliminated and furthermore the agents mayrequire removal prior to downstream use (e.g. in foodstuffs). The use ofheated water above 70° C. provides the advantage of simultaneouslypasteurising the extract (pasteurisation occurs above 73° C.) andextracting 95% of polyphenols in a single cycle.

In one embodiment, the water is used in the solvent extraction step at apressure greater than 1 bar (e.g. 2-5 bar, such as 4 bar). The provisionof water under high pressure provides the advantage of overcoming theback pressures associated with the extraction process.

In one embodiment, the extracting solvent is methanol whichadvantageously is capable of extracting 95% of polyphenols in a singlecycle.

In an alternative embodiment, the extracting solvent is acetone which isa more environmentally acceptable solvent than methanol.

In one embodiment, the extraction cycle (d) comprises a plurality ofseparate stages, suitably three stages.

In one embodiment, the first stage of the solvent extraction cycle makesuse of an extracting solvent comprising 50 to 100% methanol and 0 to 50%water, suitably 60 to 90% methanol and 10 to 40% water, for example 80%methanol and 20% water. This stage helps to extract the residualcatechin and epicatechin molecules from the powdered apples obtained instep (c).

In one embodiment, the powdered apples are hydrolysed prior to the firststage, which breaks glycoside bonds. The hydrolysis step is notnecessary for low molecular weight polyphenols because these materialsexist exclusively as non-sugar bonded polyphenols.

In one embodiment, a second stage of the solvent extraction cycle makesuse of a solvent comprising 0 to 75% ethanol and 25 to 100% water,suitably 40 to 60% ethanol and 40 to 60% water, for example 50% ethanoland 50% water. This stage assists in recovering the higher molecularweight oligomers and polymers from the apple biomass.

In one embodiment, the second stage of the extraction cycle can bepreceded by a water wash. For example, the extraction biomass maytypically be washed in a counter current extractor with water to swellthe material as a pre-stage in advance of further stage extraction. Thisstage advantageously increases the extraction availability of polymers.

In one embodiment, a third stage of the extraction cycle makes use of anextracting solvent comprising 50 to 100% acetone and 0 to 50% water,suitably 60 to 80% methanol and 20 to 40% water, for example 70% acetoneand 30% water.

In an alternative embodiment, the extraction cycle can comprise onecontinuous stage. In this case, the extraction cycle can be arranged tosequentially apply a combination of extracting solvents. As a result,the extraction sequence is: 80% methanol, 20% water, 50% methanol 50%water and 75% acetone 25% water. The continuous arrangement isbeneficially more appropriate for large scale biomass throughout whencompared with the batch extraction which is more suitable for smallerbiomass throughput and trial quantities.

The solvent extraction cycle produces an extraction liquid and a solubleapple pulp. In one embodiment, the solvent extraction cycle is repeated,suitably three times. In a further embodiment, the soluble apple pulpmay be recovered from the extraction process, dried, by methods wellknown to those skilled in the art, e.g. spray drying, thermal drying,freeze drying, etc. and packed as dehydrated apple fibre, suitably inheat sealed aluminium foil bags or containers.

In one embodiment, the isolation step (e) comprises centrifugation inaccordance with procedures known to those skilled in the art. In oneembodiment, the extraction liquid is centrifuged for 15 to 30 minutes at3000 to 9000 g. This helps to separate any remaining solid apple pulpfrom extraction liquid and eliminates the bulk biomass material prior tothe subsequent filtration step.

In one embodiment, the supernatant obtained by centrifugation issubjected to ultra-filtration. This provides the advantage ofselectively filtering out polyphenols of differing molecular weights.

In one embodiment, low molecular weight polyphenols are separated, e.g.molecules with a molecular weight of less than 600. This embodimentcreates a filtrate comprising solvent, polyphenol monomers, e.g.epicatechin and catechin (molecular weight=290), polyphenol dimers(molecular weight=580), monosaccharides and disaccharides, e.g. fructoseand sucrose (molecular weight=342).

In an alternative embodiment, molecules with a molecular weight of lessthan 300 are separated which creates a filtrate comprising solvent andpolyphenol monomers, e.g. epicatechin and catechin (molecular weight of290).

In one embodiment, the residual solvent may be removed e.g. either by asingle or multi-stage evaporation step in the presence of an inert gas(e.g. nitrogen) and recovered by condensation for recycling. In afurther embodiment, the solvent is washed with ethyl acetate to ensurethat monomers and dimers remain in the water phase solution for postprocessing prior to the final separation stage.

Therefore, the process results in two isolates, each in aqueoussolution. In one embodiment, the first isolate comprises low molecularweight polyphenols, and the second isolate comprises high molecularweight polyphenols, e.g. molecules with a molecular weight of more than600.

In a further embodiment, the isolates are both dried under vacuum at atemperature less than 30° C., e.g. less than 20° C. and optionallyfreeze-dried.

In one embodiment, there is provided a process for isolating one or morepolyphenols from an apple which comprises the steps of:

(a) removing the natural wax coating of the apple;(b) freeze-drying or freezing the apple obtained in step (a);(c) powdering the freeze-dried or frozen material obtained in step (b);(d) subjecting the material obtained in step (c) to one or more solventextraction cycles wherein the solvent is water;(e) evaporation of the water from step (d);(f) chromatographic separation of isolated polyphenols from the crudeextract prepared from step (e) by elution with ethanol;(g) evaporation of ethanol to yield the isolated polyphenols.

According to a second aspect of the invention there is provided apolyphenol containing extract obtainable by the process as hereinbeforedescribed.

It has been found that extraction of polyphenols from red-cut throughapples in accordance with the process of the invention results in a richred coloured extract having a high proportion of polyphenols (e.g.greater than 18% epicatechin). It is believed that the polyphenolswithin the extract have been fixed as anthocyanidins which are arecognised food colourant (cyanidin; E163a). This food colourant hasbeen approved for use in levels of 2.5 mg/kg body weight and the processof the invention provides cyanidin in a concentration of 1 mg/kg bodyweight. Thus, in one embodiment, there is provided a natural foodcolourant which comprises the polyphenol containing extract of theinvention. The use of the polyphenol extract as a food colouringprovides two significant advantages, safe colouration of food and theprovision of high levels of beneficial polyphenols.

Methods of Treatment

Polyphenols have been demonstrated to show numerous health benefits. Forexample, epidemiological studies have suggested that higher flavonoidintake is associated with a decreased risk of CVD, (Arts et al (2001)Epidemiology 12(6): 668-675; Sesso et al. Am J Clin Nutr 77: 1400-8;Mink et al. (2007) Am J Clin Nutr 85(3): 895-909), which may be a resultof their ability to improve endothelial function and inhibit plateletaggregation in humans (Keen et al. (2005) Am J Clin Nutr 81 (1 Suppl):298S-303S; Vita J A (2005) Am J Clin Nutr 81(Suppl): 292S-7S;Heptinstall et al. (2006) J Cardiovasc Pharmacol 47 Suppl 2: S197-205).

Thus, according to a further aspect, there is provided a polyphenolextract as hereinbefore defined for use in the prophylaxis or treatmentof cardiovascular disease or colon cancer.

In a further aspect, there is provided a use of a polyphenol extract ashereinbefore defined in the manufacture of a medicament for use in theprophylaxis or treatment of cardiovascular disease or colon cancer.

In a yet further aspect of the invention there is provided a method oftreatment or prophylaxis of cardiovascular disease or colon cancer,which comprises administration of a polyphenol extract as hereinbeforedefined.

In a yet further aspect of the invention there is provided apharmaceutical composition comprising a polyphenol extract for use inthe prophylaxis or treatment of cardiovascular disease or colon cancer.

According to a further aspect of the invention, there is provided apolyphenol extract comprising one or more low molecular weightpolyphenols for use in the prophylaxis or treatment of cardiovasculardisease.

In a further aspect, there is provided a use of a polyphenol extractcomprising one or more low molecular weight polyphenols in themanufacture of a medicament for use in the prophylaxis or treatment ofcardiovascular disease.

In a yet further aspect of the invention there is provided a method oftreatment or prophylaxis of cardiovascular disease, which comprisesadministration of a polyphenol extract comprising one or more lowmolecular weight polyphenols.

In a yet further aspect of the invention there is provided apharmaceutical composition comprising a polyphenol extract comprisingone or more low molecular weight polyphenols for use in the prophylaxisor treatment of cardiovascular disease.

In a further aspect, there is provided a polyphenol extract comprisingone or more high molecular weight polyphenols for use in the prophylaxisor treatment of colon cancer.

In a further aspect, there is provided a use of a polyphenol extractcomprising one or more high molecular weight polyphenols in themanufacture of a medicament for use in the prophylaxis or treatment ofcolon cancer.

In a yet further aspect of the invention there is provided a method oftreatment or prophylaxis of colon cancer, which comprises administrationof a polyphenol extract comprising one or more high molecular weightpolyphenols.

In a yet further aspect of the invention there is provided apharmaceutical composition comprising a polyphenol extract comprisingone or more high molecular weight polyphenols for use in the prophylaxisor treatment of colon cancer.

It will be appreciated that the term “treatment” and “treating” as usedherein means the management and care of a patient for the purpose ofcombating a condition, such as a disease or a disorder. The term isintended to include the full spectrum of treatments for a givencondition from which the patient is suffering, such as administration ofthe active compound to alleviate the symptoms or complications, to delaythe progression of the disease, disorder or condition, to alleviate orrelief the symptoms and complications, and/or to cure or eliminate thedisease, disorder or condition as well as to prevent the condition,wherein prevention is to be understood as the management and care of apatient for the purpose of combating the disease, condition, or disorderand includes the administration of the active compounds to prevent theonset of the symptoms or complications. The patient to be treated ispreferably a mammal, in particular a human being, but it may alsoinclude animals, such as dogs, cats, cows, sheep, horses and pigs.

It will also be appreciated that references herein to “polyphenolextract” are intended to include one or more polyphenols extracted froman apple in accordance with the process of the invention. This term isalso intended to include isolated polyphenols which may have beenpurified from the crude extract (e.g. by chromatographic separation).

Combination Therapies

Many diseases are treated using more than one medicament in thetreatment, either concomitantly administered or sequentiallyadministered. It is therefore within the scope of the invention to usethe polyphenol extracts of the invention in therapeutic methods for thetreatment of one of the above mentioned diseases in combination with oneanother, or as an adjunct to, or in conjunction with, other establishedtherapies normally used to in the treatment said disease. In oneembodiment, there is provided a pharmaceutical composition comprising apolyphenol extract as hereinbefore defined in combination with one ormore additional therapeutic agent.

By analogy, it is also within the scope of the invention to use thepolyphenol extracts of the invention in combination with othertherapeutically active compounds normally used in the treatment of oneof the above-mentioned diseases in the manufacture of a medicament forsaid disease.

The combination treatment may be carried out in any way as deemednecessary or convenient by the person skilled in the art and for thepurpose of this specification, no limitations with regard to the order,amount, repetition or relative amount of the compounds to be used incombination is contemplated.

Compositions

In one embodiment, the polyphenol extracts obtained by the process ofthe invention may be used in combination with other prebiotic agents,for example monosaccharides, disaccharides and polysaccharides. Thus,according to a further aspect of the invention, there is provided aprebiotic composition, comprising a polyphenol extract having one ormore high molecular weight polyphenols and one or more monosaccharide,disaccharide or polysaccharide. A suitable saccharide is oligofructosewhich has the structure shown in formula (XI):

wherein n is typically in the range 2 to 14, suitably 2 to 10, e.g. 2 to8.

In a further embodiment, the polyphenol extracts of the invention may beused in combination with conventional apple extracts, which providesvarious monosaccharides, disaccharides and polysaccharides.

When the polyphenols are used in combination with other prebiotic agentsor conventional apple extracts, the components may be administeredeither sequentially or simultaneously by any convenient route. Thiscombination provides the advantage of stimulating the micro flora, whichhelps to improve gut health, gut function, calcium absorption and immuneresponse, as well as further reducing the risk of colon cancer (Rios, L.et al., supra).

In one aspect, the invention provides a dosage form, which comprises thepolyphenol extracts and/or the combinations referred to above(hereinafter referred to as the compositions of the invention).

The compositions of the invention may conveniently be administered aloneor in combination with pharmaceutically acceptable carriers orexcipients. The compositions of the invention may be formulated withpharmaceutically acceptable carriers or diluents as well as any otherknown adjuvants and excipients in accordance with conventionaltechniques such as those disclosed in Remington: The Science andPractice of Pharmacy, 19^(th) Edition, Gennaro, Ed., Mack PublishingCo., Easton, Pa., 1995.

Suitable pharmaceutical carriers include inert solid diluents orfillers, sterile aqueous solution and various organic solvents. Examplesof solid carriers are lactose, terra alba, sucrose, cyclodextrin, talc,gelatine, agar, pectin, acacia, magnesium stearate, stearic acid andlower alkyl ethers of cellulose. Examples of liquid carriers are syrup,peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines,polyoxyethylene and water.

Similarly, the carrier or diluent may include any sustained releasematerial known in the art, such as glyceryl monostearate or glyceryldistearate, alone or mixed with a wax. The pharmaceutical compositionsformed by combining the compositions of the invention and thepharmaceutically acceptable carriers are then readily administered in avariety of dosage forms suitable for the disclosed routes ofadministration. The formulations may conveniently be presented in unitdosage form by methods known in the art of pharmacy.

Thus, in a further aspect, there is provided a dosage form comprising apolyphenol extract having one or more low molecular weight polyphenols,or a pharmaceutically acceptable salt, or prodrug thereof, and one ormore pharmaceutically acceptable carriers, excipients, or diluents foruse in the treatment of cardiovascular disease.

Thus, in a further aspect, there is provided a dosage form comprising apolyphenol extract having one or more high molecular weight polyphenols,or a pharmaceutically acceptable salt, or prodrug thereof, and one ormore pharmaceutically acceptable carriers, excipients, or diluents foruse in the treatment of colon cancer.

The dosage forms may be formulated for administration by any suitableroute such as the oral, rectal, nasal, pulmonary, topical (includingbuccal and sublingual), transdermal, intracisternal, intraperitoneal,vaginal and parenteral (including subcutaneous, intramuscular,intrathecal, intravenous and intradermal) route, the oral route beingpreferred. It will be appreciated that the preferred route will dependon the general condition and age of the subject to be treated, thenature of the condition to be treated and the active ingredient chosen.

For topical use, sprays, creams, ointments, jellies, gels, inhalants,dermal patches, implants, solutions of suspensions, etc., containing thecompounds of the present invention are contemplated. For the purpose ofthis application, topical applications shall include mouthwashes andgargles.

Compositions for oral administration include solid dosage forms, such ashard or soft capsules, tablets, troches, dragees, pills, lozenges,powders, granules, and liquid dosage forms such as solutions, emulsions,aqueous or oily suspensions, syrups and elixirs, each containing apredetermined amount of the compositions of the invention, and which mayinclude a suitable excipient. Compositions intended for oral use may beprepared according to any known method, and such compositions maycontain one or more agents selected from the group consisting ofsweetening agents, flavouring agents, colouring agents, and preservingagents in order to provide pharmaceutically elegant and palatablepreparations.

Aqueous suspensions may contain the compositions of the invention inadmixture with excipients suitable for the manufacture of aqueoussuspensions. Such excipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatidesuch as lecithin, or condensation products of an alkylene oxide withfatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample, heptadecaethyl-eneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more colouring agents,one or more flavouring agents, and one or more sweetening agents, suchas sucrose or saccharin.

Oily suspensions may be formulated by suspending the compositions of theinvention in a vegetable oil, for example arachis oil, olive oil, sesameoil or coconut oil, or in a mineral oil such as a liquid paraffin. Theoily suspensions may contain a thickening agent, for example beeswax,hard paraffin or cetyl alcohol. Sweetening agents such as those setforth above, and flavouring agents may be added to provide a palatableoral preparation. These compositions may be preserved by the addition ofan anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the compositions of theinvention in admixture with a dispersing or wetting agent, suspendingagent and one or more preservatives. Suitable dispersing or wettingagents and suspending agents are exemplified by those already mentionedabove. They may also contain buffering agents such as citrate andphosphate buffers, effervescent agents formed from carbonates, e.g.bicarbonates such as sodium or ammonium bicarbonate, and a solic acid,for example citric acid or an acid citrate salt. Additional excipients,for example, sweetening, flavouring, and colouring agents may also bepresent.

The pharmaceutical compositions of the present invention may also be inthe form of oil-in-water emulsions. The oily phase may be a vegetableoil, for example, olive oil or arachis oil, or a mineral oil, forexample a liquid paraffin, or a mixture thereof. Suitable emulsifyingagents may be naturally-occurring gums, for example gum acacia or gumtragacanth, naturally-occurring phosphatides, for example soy bean,lecithin, and esters or partial esters derived from fatty acids andhexitol anhydrides, for example sorbitan monooleate, and condensationproducts of said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions may also containsweetening and flavouring agents.

Syrups and elixirs may be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol or sucrose. Such formulations mayalso contain a demulcent, a preservative and flavouring and colouringagents. The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleaginous suspension. This suspension may beformulated according to the known methods using suitable dispersing orwetting agents and suspending agents described above. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent,for example as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conveniently employed as solvent or suspending medium. For thispurpose, any bland fixed oil may be employed using synthetic mono- ordiglycerides. In addition, fatty acids such as oleic acid find use inthe preparation of injectables.

In a further embodiment, the polyphenol extracts can be dried, forexample spray-dried or dried under vacuum at a temperature less than 30°C., e.g. less than 20° C. and optionally freeze-dried, and formulatedinto a solid dosage form.

Tablets may contain the active ingredient in admixture with non-toxicpharmaceutically acceptable excipients which are suitable for themanufacture of tablets. These excipients may be for example, inertdiluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example corn starch or alginic acid; binding agents, for example,starch, gelatine or acacia; and lubricating agents, for examplemagnesium stearate, stearic acid or talc.

The tablets may be uncoated or they may be coated by known techniques todelay disintegration and absorption in the gastrointestinal tract andthereby provide a sustained action over a longer period.

Formulations for oral use may also be presented as hard gelatinecapsules where the active ingredient is mixed with an inert soliddiluent, for example, calcium carbonate, calcium phosphate or kaolin, ora soft gelatine capsules wherein the active ingredient is mixed withwater or an oil medium, for example peanut oil, liquid paraffin, orolive oil.

Dosage forms for parenteral administration include sterile aqueous andnon-aqueous injectable solutions, dispersions, suspensions or emulsionsas well as sterile powders to be reconstituted in sterile injectablesolutions or dispersions prior to use. Such aqueous solutions should besuitably buffered if necessary and the liquid diluent first renderedisotonic with sufficient saline or glucose. The aqueous solutions areparticularly suitable for intravenous, intramuscular, subcutaneous andintraperitoneal administration. The sterile aqueous media employed areall readily available by standard techniques known to those skilled inthe art. Depot injectable formulations are also contemplated as beingwithin the scope of the present invention.

The compositions for rectal administration of the compounds may also bein the form of suppositories. These compositions can be prepared bymixing the compositions of the invention with a suitable non-irritatingexcipient which is solid at ordinary temperatures but liquid at therectal temperature and will thus melt in the rectum to release thecompositions of the invention. Such materials include cocoa butter andpolyethylene glycols, for example.

The compounds of the invention, or compositions thereof, will generallybe used in an amount effective to achieve the intended result, forexample in an amount effective to treat or prevent the particulardisease being treated. The compound(s) may be administeredtherapeutically to achieve therapeutic benefit. By therapeutic benefitis meant eradication or amelioration of the underlying disorder beingtreated and/or eradication or amelioration of one or more of the systemsassociated with the underlying disorder. Therapeutic benefit alsoincludes halting or slowing the progression of the disease, regardlessof whether improvement is realised.

The exact dosage will depend upon the frequency and mode ofadministration, the sex, age, weight and general condition of thesubject treated, the nature and severity of the condition treated andany concomitant diseases to be treated and other factors evident tothose skilled in the art. Determination of the effective dosage is wellwithin the capabilities of those skilled in the art.

When the polyphenol extract is used in combination with a secondtherapeutic agent active against the same disease state the dose of eachcompound may differ from that when the compound is used alone.Appropriate doses will be readily appreciated by those skilled in theart.

In a further aspect, the invention provides a dietary composition, e.g.a drink, such as a fruit juice, sports drink, yoghurt drink, a milkdrink, tea and the like or a solid foodstuff, e.g. a food snack bar,such as a fruit bar, nut bar and cereal bar, a cereal, a dessert, achocolate (e.g. milk and dark) bar and the like, which comprises thepolyphenol extracts and/or the combinations referred to above.

In one embodiment, the dietary composition may additionally compriseother nutrients, e.g. vitamins, minerals, and prebiotics, such asoligofructose and apple fibres, and probiotics.

In one embodiment, the dietary composition is formulated as a liquiddosage form as hereinbefore defined. In a further embodiment, the liquiddosage form may additionally comprise thickeners, tonicity adjustingagents and buffering agents.

Examples of suitable tonicity adjusting agents include sugars and sodiumchloride, which can be used to provide a solution of a particularstrength, for example and isotonic solution. Examples of suitablebuffering agents include citrates and phosphates.

In a further embodiment, the foodstuff comprises the polyphenol extractas hereinbefore defined. In a further embodiment, the polyphenol extractmay be admixed with one or more ingredients, for example dried fruit,nuts and cereals.

Thus, in a further aspect, there is provided a dietary composition ashereinbefore defined comprising a polyphenol extract having one or morelow molecular weight polyphenols for use in the prophylaxis or treatmentof cardiovascular disease.

In a further aspect, there is provided a dietary composition ashereinbefore defined comprising a polyphenol extract having one or morelow molecular weight polyphenols in the manufacture of a medicament foruse in the prophylaxis or treatment of cardiovascular disease.

In a yet further aspect of the invention there is provided a method oftreatment or prophylaxis of cardiovascular disease, which comprisesadministration of a dietary composition as hereinbefore definedcomprising a polyphenol extract having one or more low molecular weightpolyphenols.

Thus, in a further aspect, there is provided a dietary composition ashereinbefore defined comprising a polyphenol extract having one or morehigh molecular weight polyphenols for use in the prophylaxis ortreatment of colon cancer.

In a further aspect, there is provided a dietary composition ashereinbefore defined comprising a polyphenol extract having one or morehigh molecular weight polyphenols in the manufacture of a medicament foruse in the prophylaxis or treatment of colon cancer.

In a yet further aspect of the invention there is provided a method oftreatment or prophylaxis of colon cancer, which comprises administrationof a dietary composition as hereinbefore defined comprising a polyphenolextract having one or more high molecular weight polyphenols.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 demonstrates a schematic arrangement of the process of theinvention.

FIGS. 2-13 demonstrate the results of the analysis of the effect ofsolvent extraction temperature upon epicatechin extraction.

FIGS. 14 and 15 demonstrate the results of the methanol extractionanalysis.

FIG. 16 demonstrates the results of the ethanol extraction analysis.

FIGS. 17 and 18 demonstrate the results of the cardiovascular study withepicatechin extracts.

The invention will now be illustrated by the following non-limitingExamples.

EXAMPLES General Procedures

Approximately 6 g of freeze-dried apple was weighed into a beaker andthe weight recorded. To this 15 ml of deionised, distilled, degassedwater is added to form a thick paste, which is then transferred to anextraction cell which has been part filled with deionised and deaeratedwater. The remaining cell volume was filled with water, sealed andplaced into the oven of the extraction system (which is shown in FIG.1). Water was pumped in until a stable pressure of approx 4 bar wasachieved. The oven was then set to the desired temperature and allowedto equilibrate for 30 minutes prior to the start of the samplecollection phase.

After 30 minutes with water flow at a fixed rate, samples were collectedover a two-hour period, which were sampled and analysed by HPLC. Theconcentration of (−)-epicatechin in solution was then calculated andused to assess the rate of extraction over a range of temperatures.

HPLC Method

Column: Waters RP18 (C₁₈ stationary phase 4.6×150 mm, 3.5 μm particlesize)Grad column: Waters RP18 (4.6×50 mm, 3.5 μm particle size)Oven temperature: 30° C.Binary mobile phase: Component A. Water containing 2% acetic acid.

-   -   Component B. Methanol containing 0.1% acetic acid

TABLE 1 Gradient elution profile Elapsed time Gradient type % A % B(min) Initial 85 15 0 Linear 55 45 25 Linear 15 85 26 Hold 15 85 35Linear 85 15 37 Hold 85 15 40 Post run time 85 15 5

Example 1 Effect of Solvent Extraction Temperature Upon EpicatechinExtraction

The extraction analysis was performed on 6 different temperatures asshown in Table 2.

TABLE 2 Extraction Conditions Water flow rate Test I.D. Temperature (°C.) (ml/min) A 20 (Ambient) 100 ml static volume B 50 1.0 C 75 1.0 D 1001.0 E 125 1.0 F 150 1.0

(A) Extraction at 20° C.

10.001 g of apple powder were placed in a beaker with 100 ml water,mixed well covered with sealing film and left for 2 hours. 1 ml of theresulting solution was then filtered through a 0.45 μm syringe filterinto a 2 ml auto sampler vial to which was added 50 μm of internalstandard solution of epicatechin (obtained from Sigma). The results ofthe chromatography are shown in FIG. 2 and once analysed the solutiongave rise to a concentration of 825.28 mg epicatechin/Kg of applepowder.

(B) Extraction at 50° C.

6.137 g of apple powder were extracted in accordance with the “GeneralProcedures” previously described. 1 ml of each of the resultingsolutions was then filtered through a 0.45 μm syringe filter into a 2 mlauto sampler vial to which was added 50 μl of internal standard solutionof epicatechin (obtained from Sigma). The results are presented in Table3.

TABLE 3 Results of 50° C. Extraction (−)- epicatechin Total (−)- % cumulExtract vol (mg) in epicatechin (−)- (ml) volume extracted (mg)epicatechin 10 0.247 0.247 2.0 15 0.062 0.308 2.5 25 0.577 0.885 7.2 354.597 5.482 44.5 45 1.498 6.980 56.6 55 2.554 9.534 77.4 65 1.607 11.14190.4 75 0.689 11.830 96.0 85 0.287 12.117 98.3 95 0.147 12.264 99.5 1050.058 12.322 100.0 110 0.000 12.322 100.0

The cumulative results of the 50° C. extraction are shown in FIG. 3 andthe results of the chromatography are shown in FIG. 4.

The results of this analysis obtained 2.01 mg of epicatechin per gram ofapple powder (equivalent to 2007.85 mg/kg):

Sample (g) mg/g mg/kg 50° C. 6.137 2.01 2007.85

(C) Extraction at 75° C.

6.595 g of apple powder were extracted in accordance with the “GeneralProcedures” previously described. 1 ml of each of the resultingsolutions was then filtered through a 0.45 μm syringe filter into a 2 mlauto sampler vial to which was added 50 μl of internal standard solutionof epicatechin (obtained from Sigma). The results are presented in Table4.

TABLE 4 Results of 75° C. Extraction (−)- epicatechin Total (−)- % cumulExtract vol (mg) in epicatechin (−)- (ml) volume extracted (mg)epicatechin 10 3.097 3.097 16.8 20 4.926 8.023 43.6 30 2.981 11.004 59.840 2.045 13.049 70.9 51 1.553 14.602 79.4 60 1.025 15.627 84.9 70 0.84116.468 89.5 80 0.683 17.151 93.2 93 0.663 17.814 96.8 100 0.233 18.04798.1 110 0.222 18.270 99.3 120 0.131 18.400 100.0

The cumulative results of the 75° C. extraction are shown in FIG. 5 andthe results of the chromatography are shown in FIG. 6.

The results of this analysis obtained 2.79 mg of epicatechin per gram ofapple powder (equivalent to 2790.02 mg/kg):

Sample (g) mg/g mg/kg 75° C. 6.595 2.79 2790.02

(D) Extraction at 100° C.

6.0962 g of apple powder were extracted in accordance with the “GeneralProcedures” previously described. 1 ml of each of the resultingsolutions was then filtered through a 0.45 μm syringe filter into a 2 mlauto sampler vial to which was added 50 μl of internal standard solutionof epicatechin (obtained from Sigma). The results are presented in Table5.

TABLE 5 Results of 100° C. Extraction (−)- epicatechin Total (−)- %cumul Extract vol (mg) in epicatechin (−)- (ml) volume extracted (mg)epicatechin 11 1.126 1.126 6.9 21 4.089 5.215 31.9 31 3.819 9.034 55.341 2.806 11.840 72.5 52 1.884 13.724 84.0 61 1.087 14.811 90.6 71 0.67015.481 94.7 81 0.429 15.910 97.4 91 0.214 16.123 98.7 102 0.162 16.28599.7 111 0.057 16.342 100.0 121 0.000 16.342 100.0 161 0.000 16.342100.0

The cumulative results of the 100° C. extraction are shown in FIG. 7 andthe results of the chromatography are shown in FIG. 8.

The results of this analysis obtained 2.68 mg of epicatechin per gram ofapple powder (equivalent to 2680.71 mg/kg):

Sample (g) mg/g mg/kg 100° C. 6.0962 2.68 2680.71

(E) Extraction at 125° C.

6.2038 g of apple powder were extracted in accordance with the “GeneralProcedures” previously described. 1 ml of each of the resultingsolutions was then filtered through a 0.45 μm syringe filter into a 2 mlauto sampler vial to which was added 50 μl of internal standard solutionof epicatechin (obtained from Sigma). The results are presented in Table6.

TABLE 6 Results of 125° C. Extraction (−)- epicatechin Total (−)- %cumul Extract vol (mg) in epicatechin (−)- (ml) volume extracted (mg)epicatechin 15 4.003 4.003 31.2 25 4.505 8.508 66.2 35 1.910 10.418 81.145 1.130 11.549 89.9 55 0.513 12.061 93.9 65 0.305 12.367 96.3 75 0.17312.540 97.6 85 0.099 12.639 98.4 95 0.087 12.725 99.1 106 0.056 12.78199.5 115 0.032 12.813 99.8 125 0.030 12.843 100.0 165 0.000 12.843 100.0

The results of the chromatography are shown in FIG. 9.

The results of this analysis obtained 2.07 mg of epicatechin per gram ofapple powder (equivalent to 2070.21 mg/kg):

sample (g) mg/g mg/kg 125° C. 6.2038 2.07 2070.21

(F) Extraction at 150° C.

6.6651 g of apple powder were extracted in accordance with the “GeneralProcedures” previously described. 1 ml of each of the resultingsolutions was then filtered through a 0.45 μm syringe filter into a 2 mlauto sampler vial to which was added 50 μl of internal standard solutionof epicatechin (obtained from Sigma). The results are presented in Table7.

TABLE 7 Results of 150° C. Extraction (−)- epicatechin Total (−)- %cumul Extract vol (mg) in epicatechin (−)- (ml) volume extracted (mg)epicatechin 16 1.061 1.061 42.9 29 0.901 1.962 79.4 39 0.245 2.208 89.349 0.120 2.327 94.1 59 0.101 2.429 98.2 69 0.044 2.473 100.0 79 0.0002.473 100.0 93 0.000 2.473 100.0 103 0.000 2.473 100.0 116 0.000 2.473100.0 126 0.000 2.473 100.0 136 0.000 2.473 100.0

The cumulative results of the 100° C. extraction are shown in FIG. 10and the results of the chromatography are shown in FIG. 11.

The results of this analysis obtained 0.37 mg of epicatechin per gram ofapple powder (equivalent to 371.00 mg/kg):

sample (g) mg/g mg/kg 150° C. 6.6651 0.37 371.00

(G) Summary of Results

The data obtained from each of extractions (A)-(F) were overlaid and areshown in FIG. 12 which demonstrates a comparison in apparent extractionrate at a given temperature. FIG. 13 shows the effectiveness of each ofthe extraction temperatures for extraction of epicatechin (peakeffectiveness was shown to be 83%). From FIG. 13 it can be seen thatabove 75° C., the overall concentration of epicatechin extracted falls.It is possible that the reduced effectiveness may be caused byisomerisation (epimerisation) of epicatechin to catechin at highertemperatures.

Example 2 Detection of Catechin in Extracts

In order to test the possibility that epicatechin was isomerising tocatechin at higher temperatures, a test solution of 0.688 mg/mlepicatechin was produced and treated as follows: 950 μl of epicatechinwas transferred to an auto sampler vial, which was flushed with nitrogenand sealed. The vial was then placed in an oven at a given temperature,for a set time period. The vial was then cooled, the seal removed and50/41 of internal standard added before analysis. Tests were carried outat 50, 75, 100 and 125° C. for 15 and 30 minutes. Tests were not carriedout at 150° C. as the vials are not capable of withstanding the pressurethat would be generated.

The concentrations of epicatechin and catechin were determined for eachtest solution and are shown in Table 8.

TABLE 8 (−)-Epicatechin isomerisation results Epicatechin Catechin % %Other* mg/ml remain mg/ml of start Other % of start Start 0.688 100.000.000 0.00 0.000 0.00  50° C. 15 min 0.643 93.53 0.007 1.07 0.037 5.40 50° C. 30 min 0.601 87.40 0.012 1.81 0.074 10.79  75° C. 15 min 0.55079.92 0.065 9.49 0.073 10.59  75° C. 30 min 0.441 64.07 0.112 16.280.135 19.65 100° C. 15 min 0.254 36.97 0.321 46.68 0.113 16.36 100° C.30 min 0.164 23.88 0.344 50.05 0.179 26.07 125° C. 15 min 0.156 22.710.340 49.36 0.192 27.94 125° C. 30 min 0.139 20.27 0.300 43.60 0.24936.13 *note calculated from 100 − (epicatechin + catechin).

The rapid isomerisation of (−)-epicatechin at temperatures above 100° C.indicated that these temperatures are unsuitable for further study, andthat the overall time at which the sample is kept at elevatedtemperatures should be maintained as short as possible.

Example 3 Methanol Extraction Analysis

Triplicate extractions were performed with methanol on approximately 1 gof apple powder extracted in accordance with the “General Procedures”previously described. The apple sample was weighed into a 60 mlcentrifuge tube and 25 ml of methanol added. The samples were shaken andplaced in an oven set at 55° C. for 20 minutes and allowed to extract,the samples were re-shaken every 5 minutes in order to maximise contact.Once extracted the samples were removed from the oven and allowed tocool for 30 minutes. 1 ml of each solution was then filtered through a0.45 μm syringe filter into a 2 ml auto sampler vial to which was added50 μl of internal standard solution. The results are demonstrated inTable 9.

TABLE 9 Results of Methanol Extraction mg in Sample mg/ml Vol tot volMeth 1 0.32 25 8.01 Meth 2 0.35 25 8.73 Meth 3 0.38 25 9.58 Average 8.77sample (g) mg/g mg/kg Meth 1 2.0034 4.00 4000.29 Meth 2 2.0443 4.274269.64 Meth 3 2.2301 4.30 4295.73 Average 4188.55

The results of the chromatography are shown in FIG. 14.

The spectra of the (−)-epicatechin peak obtained in FIG. 14 was examinedin order to determine if there was any co-elution of other substanceswhich were not picked up at the data collection wavelength of 280+/−10nm. The amplification of this peak is shown in FIG. 15. The two spectrawere found to be identical to that produced by the (−) epicatechinstandard used for quantification. This suggests that the main peak andshoulder are composed of very similar materials, which is evidence thatthe shoulder may be the (+) isomer of epicatechin. The lack of othertraces in the spectra indicated that no other materials are co-elutingat this point in the chromatogram.

Example 4 Isolation of Polyphenols by Chromatography

Small-scale tests were performed using C18-E SPE material and twocommercially available ion exchange resins, Amberlite XAD-16 andPurolite MN100.

A test solution containing 9.18 mg of epicatechin in 10 mls of water(0.918 mg/ml) was applied to columns containing 1 g of each of the threetest sorbents. The water post treatment was assessed for the presence ofepicatechin and it was found that the XAD16 contained 0.261 mg/mlmeaning that out of the total applied to the column only 71.6% had beenretained. The MN100 and C18 showed no epicatechin present in the treatedwater. Each of the test sorbents were then eluted with ethanol and thesolvent analysed to determine the amount of epicatechin recovered.

The XAD 16 wash was found to contain 5.22 mg of epicatechin giving atotal of 7.83 mg (85.3%) of the total added to the sorbent. Leaving 1.35mg (16.5%) not recovered.

The MN 100 wash was found to contain 4.37 mg of epicatechin giving atotal of 4.37 mg (47.6%) of the total added to the sorbent. Leaving 4.81mg (52.4%) not recovered.

The C18 wash was found to contain 9.03 mg of epicatechin giving a totalof 9.03 mg (98.4%) of the total added to the sorbent. Leaving 0.15 mg(1.6%) not recovered.

From the above it can be seen that either the Amberlite XAD 16 or C18-Ecan be used, however if the XAD-16 is used it would require a muchlarger bed volume to achieve the same sample loading characteristics.Based on the above data and in order to maximise product return andsample loading, C18-E was found to have the best characteristics forisolating polyphenols from aqueous solution as shown in Table 10:

TABLE 10 Results of Chromatographic Separation with C18 C18 test VolumeConc Total (mg) data (ml) (mg/ml) added Recovery % Feed 10.0 0.918 9.18Recovered 3.0 3.01 9.03 98.4

The cumulative results of the extraction with C18 can be seen in FIG.16.

Example 5 Ethanol Extraction Analysis

Seven portions of apple powder (approx 50 g) were extracted at a ratioof 4:1 with ethanol at 50° C. The resulting collected ethanol wasfiltered through a glass fibre filter before being evaporated to collectany solid material available. In total, 377.3 g of apple powder wasextracted using 1505 g of ethanol, a ratio of 4:1.

During the evaporation process a quantity of solid material was seen tofall out of solution, this material was recovered by centrifuging thesolution. The material collected, 14.6 g of a white sticky solid wasanalysed and found to contain minute traces of polyphenols, which areprobably the result of traces of ethanol trapped in the material.

The remainder of the ethanol was recovered under vacuum at 60° C. and asticky red semi solid material was recovered weighing 28.9 g. Thismaterial was not as expected, a powdery solid. It was probable that thisfinal evaporate contained residues of ethanol, some water, lipids andproteins along with the polyphenols. In order to remove these unwantedsubstances from the phenols this solid was re-dissolved in water so thatit could be passed through a C18-E SPE tube to recover the polyphenolrich fraction.

When the final evaporate was dissolved in water, a layer of non solublematerial was seen, when filtered and dried, this material was found tobe a solid that contained trace levels of phenols. When the waterextract had been passed through the C18-E, the water was analysed andfound to contain no epicatechin. The C18-E was eluted with four timesthe bed volume of ethanol. The ethanol once evaporated gave rise to1.976 g of red powder, which when analysed was found to contain 18.3%epicatechin.

As the extract had been manipulated many times during the process to getto the powder it was decided to repeat the process without collectingdifferent fractions and proceed as directly as possible to productcollection.

The following process was therefore conducted:

1). 338 g of freeze dried apple powder was placed in a 2.5 L glassbottle and extracted with 1045 g of ethanol (ratio of 3:1) for 15minutes at 60° C.2). The resulting mixture was hot filtered by vacuum and the ethanolrecovered. The apple powder, which still contained some ethanol, wasplaced back into the extraction bottle and sealed.3). The collected extract was evaporated by 50% and any non-solublematerial removed.4). The remaining volume was further reduced the to around 15% of theinitial level for overnight storage.5). The recovered ethanol was then added back to the apple powder forre-extraction overnight at ambient again the ratio was 3:1.6). Following overnight soaking, steps 2 to 4 were repeated.7). The two concentrated ethanol extracts were combined and evaporateduntil a sticky red semi-solid was produced.8). 400 ml of hot (50° C.) de-aerated water was added to the stickysolid to dissolve the polyphenols.9). The aqueous solution was cooled and filtered to remove anynon-soluble material (fats).10). Four, C18-E, 20 g giga tubes were prepared in accordance with themanufacturers instructions prior to use. The C18 bed was first wettedwith ethanol then exchanged with four times the bed volume, 240 ml ofwater to condition the sorbent.11). The aqueous solution was processed through the C18 tubes to removethe polyphenol rich fraction from solution before washing the bed withwater to remove any non bound material.12). The sorbent bed was then eluted with 240 ml of ethanol, andcollected.13). The ethanol was evaporated to dryness and the solid “powder”collected.14). A sample of between 2 and 5 mg was analysed for its epicatechincontent.

From the initial 338 g of apple powder processed a total of 6.757 g ofred powder was obtained which has an epicatechin content of 12.1%. Thesolids recovered represent 2% of the initial apple powder mass. Whenequated to a mg/Kg equivalent of dry apple powder the recovered solidsgive a figure of 2427.1 mg/Kg.

A comparative extract using 1 g of apple powder and 10 ml methanol gavea figure of 4663 mg/Kg. In order to test if the low recovery figureabove was due to low solubility of epicatechin in ethanol a series oftests were performed using 10, 20 and 30:1 w/w. The results of which canbe seen in Table 11:

TABLE 11 Comparison of ethanol extraction with methanol Sample mg/ml mlstotal mg Sample mg/g mg/kg Methanol 0.52 10 5.20 1.115 4.66 4663.7Ethanol 10 ml 0.345 10 3.45 1.206 2.86 2862.4 Ethanol 20 ml 0.244 204.87 1.029 4.74 4736.3 Ethanol 30 ml 0.210 30 6.31 1.241 5.09 5085.5

The data in Table 11 implies that epicatechin has a lower solubility inethanol than in methanol, but by increasing the ratio comparablesolubility's and hence higher yields can be achieved.

Example 6 Cardiovascular Study with Epicatechin Extracts

10 healthy volunteers were recruited to test the effectiveness of theepicatechin extract obtained from the process of the invention as ameans of improving the vascular tone and therefore cardiovascularfunction. 5 volunteers were given a placebo fruit drink and 5 volunteerswere provided with the same test drink, but with 1 mg/kg body weight(based on (−)-epicatechin units) added. Vascular tone was continuouslyquantified by the reflective index obtained by non-invasive digitalphotoplethysmography and an algorithm for continuous,investigator-independent, automatic analysis of digital volume pulse.The data were presented as relative Stiffness Index SI (m/s) andcorrelated with vascular age. Pulse waves were measured by thetransmission of red and infrared light through the finger pulp. Thelocal minimum of the first derivative was determined and thecorresponding turning point (=inflection point) of the pulse wave wasthereby defined. The reflective index was calculated from the mean ofthe third to the seventh data point after the turning point (=inflectionpoint).

FIGS. 17 and 18 show the results of a 2.5 hour trial. These figuresindicate that the arterial stiffness index (SI; FIG. 17) and theVascular Age (FIG. 18) of the group (n=10) consuming the test drinkincluding the extract was statistically improved (i.e. reduced) whencompared to the control.

1.-11. (canceled)
 12. A polyphenol containing extract prepared by aprocess comprising the steps of: (a) removing the coating of the fruit;(b) freeze-drying or freezing the fruit obtained in step (a); (c)optionally powdering the freeze-dried or frozen material obtained instep (b); (d) subjecting the material obtained in step (b) or step (c)to one or more solvent reaction cycles; and optionally thereafter (e)isolating one or more polyphenols from the liquid extract obtained instep (d). 13.-17. (canceled)
 18. A polyphenol containing extractprepared by the method comprising the steps of: (a) removing the naturalwax coating of the apple; (b) freeze-drying or freezing the appleobtained in step (a); (c) powdering the freeze-dried or frozen materialobtained in step (b); (d) subjecting the powdered material obtained instep (c) to one or more solvent extraction cycles wherein the solvent iswater; (e) evaporation of the water from step (d); (f) chromatographicseparation of isolated polyphenols from the crude extract prepared fromstep (e) by elution with ethanol; and (g) evaporation of ethanol toyield the isolated polyphenols.
 19. A food colourant which comprises thepolyphenol containing extract of claim 12 or
 18. 20. A phenol containingextract of claim 12, wherein the fruit is an apple.
 21. A phenolcontaining extract of claim 12, wherein the fruit is a “red-cut through”apple.
 22. A phenol containing extract of claim 12, wherein thepolyphenol comprises epicatechin.
 23. A phenol containing extract ofclaim wherein step (a) further comprises a washing step using a suitablesolvent, such as ethanol, methanol or acetone.
 24. A phenol containingextract of claim 12, wherein step (c) comprises powdering in aconventional food processor in the presence of liquid nitrogen.
 25. Aphenol containing extract of claim 12, wherein the extracting solvent instep (d) is water.
 26. A phenol containing extract of claim 23, whereinthe water is deaerated water.
 27. A phenol containing extract of claim25 or 26, wherein the water is heated to a temperature between 65° C.and 80° C.
 28. A phenol containing extract of claim 25 or 26, whereinthe water is at a pressure greater than 1 bar and less than or equal to5 bar.
 29. A pharmaceutical composition comprising the polyphenolcontaining extract of claim 12 or
 18. 30. A method for the prophylaxisor treatment of cardiovascular disease or colon cancer comprisingadministering to a patient a polyphenol containing extract of claim 12or 18.